Methods for the Characterization of Microorganisms on Solid or Semi-Solid Media

ABSTRACT

The present invention relates to methods and systems for scanning, detecting, and monitoring microorganisms on solid or semi-solid media using intrinsic fluorescence (IF) measurements. The methods are further directed to detection, characterization and/or identification of microorganisms on a solid or semi-solid media using intrinsic fluorescence (IF) measurements that are characteristic of said microorganisms.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional PatentApplication No. 61/122,925, entitled, “Methods for Characterization ofMicroorganisms on Solid or Semi-Solid Media”, filed Dec. 16, 2008, whichis incorporated herein.

FIELD OF THE INVENTION

The present invention relates to methods and systems for detecting,monitoring, characterizing, and/or identifying microorganisms on solidor semi-solid media.

BACKGROUND OF THE INVENTION

Microorganisms isolated for the purpose of clinical diagnostics, as wellas those isolated to monitor contamination of foodstuffs, medicaltissues, or the environment, often need to be characterized in order todetermine the appropriate response to the presence of the organismsfound. Traditional automated phenotypic identification assays, such asthe Vitek®, Phoenix™, and Microscan® systems, or manual phenotypic testssuch as API, require that microorganisms be in an appropriate growthphase and free of interfering media and blood products in order toprovide robust results. These systems use colonies grown for 16-24 hourson plated media, after which standardized suspensions are made from thecolonies, and then the actual characterization tests require a further4-24 hours of incubation to complete.

Optical spectroscopy methods, such as intrinsic fluorescence (IF),infrared spectroscopy (FTIR), or Raman spectroscopy, have the potentialto allow for identification of microorganisms very quickly, but haveonly been demonstrated to work with “clean” microorganism suspensions.Publications have described IF methods for microbial characterizationwith only very limited organism sets, or that required additionalmeasures, such as specific binding events, to allow functionalcharacterization. Direct examination of microorganisms on growth mediumhas been considered problematic due to the assumed large contribution ofthe medium itself to the spectroscopic pattern.

The present invention overcomes the problems in the art by providingmethods that can discriminate between microorganisms spectroscopicallyinterrogated directly on fluorescent solid and/or semi-solid growthmedia, including highly fluorescent media.

SUMMARY OF THE INVENTION

The present invention provides methods for detecting, monitoring,characterizing, and/or identifying microorganisms on solid and/orsemi-solid media. Characterization encompasses the broad categorizationor classification of microorganisms as well as the actual identificationof a single species. As used herein “identification” means determiningto which family, genus, species, and/or strain a previously unknownmicroorganism belongs to. For example, identifying a previously unknownmicroorganism to the family, genus, species, and/or strain level. Themethods disclosed herein allow the detection, characterization and/oridentification of microorganisms more quickly than prior techniques,resulting in faster diagnoses (e.g., in a subject having or suspected ofhaving an infection) and identification of contaminated materials (e.g.,foodstuffs and pharmaceuticals). The steps involved in the methods ofthe invention can be carried out in a short time frame to produceclinically relevant actionable information. In certain embodiments, fastgrowing organisms can be detected and identified in just a few hours.Slower growing organisms can be detected and identified more quicklythan with prior techniques, providing results in a useful timeframe. Theidentification/characterization step alone can be carried in a fewminutes or less. The methods also permit detecting, monitoring,characterizing, and/or identifying multiple types of microorganisms(e.g., different classes and/or species) simultaneously (e.g., in mixedcultures). Advantageously, in some embodiments, the methods of theinvention can be performed in situ without destruction of the colony,thereby preserving the colony for further tests or uses. Additionally,the methods of the invention can be partially or fully automated,thereby reducing the risk of handling infectious materials and/orcontaminating the samples.

A first aspect of the invention relates to methods of characterizingand/or identifying a microorganism on a solid or semi-solid medium,comprising:

(a) interrogating one or more colonies on a solid or semi-solid mediumto produce intrinsic fluorescence (IF) measurements characteristic of amicroorganism in the colony; and(b) characterizing and/or identifying the microorganism in the colonybased on intrinsic fluorescence (IF) measurements.

Another aspect of the invention relates to methods of detecting andcharacterizing a microorganism on a solid or semi-solid medium,comprising:

(a) scanning a medium, known to contain, or that may contain one or moremicroorganism colonies to locate said colonies present on the medium;(b) interrogating one or more colonies located during step (a) toproduce intrinsic fluorescence (IF) measurements characteristic of amicroorganism in the colony; and(c) detecting, characterizing and/or identifying the microorganism inthe colony based on said intrinsic fluorescence (IF) measurements.

A further aspect of the invention relates to methods of characterizingand/or identifying a microorganism in a sample, comprising:

(a) growing a microorganism present in the sample on a solid orsemi-solid medium to produce at least one colony;(b) interrogating one or more colonies on the medium to produceintrinsic fluorescence (IF) measurements characteristic of themicroorganism; and(c) characterizing and/or identifying the microorganism in the colonybased on the produced measurements.

An additional aspect of the invention relates to methods of detectingthe presence of a microorganism in a sample, comprising:

(a) obtaining a sample known to contain or that may contain amicroorganism;(b) growing a microorganism present in the sample on a solid orsemi-solid medium; and(c) locating any colonies present on the medium by conducting apoint-by-point scanning of said solid or semi-solid medium to produceintrinsic fluorescence (IF) measurements;wherein the presence of one or more colonies as located by the producedmeasurements indicates that a microorganism is present in the sample.

In one embodiment, the invention relates to a system for detecting,characterizing and/or identifying a microorganism on a solid orsemi-solid medium, the system comprising a spectrophotometer andfocusing optics, such as a lens system or a microscope. In otherembodiments, the system further comprises a mechanism for scanning thesurface of the medium and/or a mechanism for controlling the environmentof (e.g., incubating) the medium.

In another embodiment, a colony can be interrogated to producemeasurements which can be used to detect, characterize and/or identifythe microorganisms of the colony (e.g., the colony can be interrogatedusing spectroscopy). The microorganisms can be characterized and/oridentified by comparing the measurements (e.g., the spectrum) of thecolony to similar measurements (e.g., spectrum or spectra) taken ofknown microorganisms. In another embodiment, the colony can beinterrogated non-invasively (e.g., within a sealed plate). The abilityto characterize and/or identify the microorganisms contained in a colonydirectly (e.g., within a sealed plate) without further handling enhancesthe safety of microbial identification.

In yet another embodiment, the spectroscopic interrogation is based onintrinsic characteristics of the microorganisms (e.g., intrinsicfluorescence). In other embodiments, the spectroscopic interrogation isbased in part on signals obtained from additional agents that are addedduring the methods of the invention and interact with specificmicroorganisms or groups of microorganisms.

In another embodiment, the methods further comprise a step of recoveringthe colony, resuspending the colony and performing furtheridentification and/or characterization tests (e.g., drug resistance,virulence factors, antibiogram).

The present invention is explained in greater detail in the figuresherein and the description set forth below.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1D show spectra from uninoculated blood agar plates (BAP) withno membrane (A) or BAP with Pall Metricel Black gridded polyethersulfonemembrane (Pall) (B), Whatman black mixed ester membrane (WME) (C), orWhatman track-etched polycarbonate black membrane (WPC) (D) laid acrossthe surface of the medium.

FIGS. 2A-2C show spectra from colonies on WME membrane over BAP obtainedfrom EC3 (A) and SA1 (B), and the results of subtracting the EC3spectrum from the SA1 spectrum (C).

FIGS. 3A-3D show spectra from colonies on BAP without a membraneobtained from EC1 (A), SA1 (B), EF1 (C), and PA1 (D).

FIGS. 4A-4F show three dimensional plots of the point-by-point IF searchscans of run F, where height equals fluorescence intensity. The plotsshow measurements taken at 6 h (A), 8 h (B), 10 h (C), 12 h (D), 16 h(E), and 24 h (F).

FIGS. 5A-5B show a close-up image of the BAP from run F after 24 h (A),and a contour plot of fluorescence intensity from the search scan at 12h showing corresponding colony locations (B).

DETAILED DESCRIPTION OF THE INVENTION

The present invention can be embodied in different forms and should notbe construed as limited to the embodiments set forth herein. Rather,these embodiments are provided so that this disclosure will be thoroughand complete, and will fully convey the scope of the invention to thoseskilled in the art. For example, features illustrated with respect toone embodiment can be incorporated into other embodiments, and featuresillustrated with respect to a particular embodiment can be deleted fromthat embodiment. In addition, numerous variations and additions to theembodiments suggested herein will be apparent to those skilled in theart in light of the instant disclosure, which do not depart from theinstant invention.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. The terminology used in thedescription of the invention herein is for the purpose of describingparticular embodiments only and is not intended to be limiting of theinvention.

DEFINITIONS

As used herein, “a,” “an,” or “the” can mean one or more than one. Forexample, “a” cell can mean a single cell or a multiplicity of cells.

Also as used herein, “and/or” refers to and encompasses any and allpossible combinations of one or more of the associated listed items, aswell as the lack of combinations when interpreted in the alternative(“or”).

Furthermore, the term “about,” as used herein when referring to ameasurable value such as an amount of a compound or agent, dose, time,temperature, and the like, is meant to encompass variations of ±20%,±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified amount.

As used herein, the term “microorganism” is intended to encompassorganisms that are generally unicellular, which can be multiplied andhandled in the laboratory, including but not limited to, Gram-positiveor Gram-negative bacteria, yeasts, molds, parasites, and mollicutes.Non-limiting examples of Gram-negative bacteria of this inventioninclude bacteria of the following genera: Pseudomonas, Escherichia,Salmonella, Shigella, Enterobacter, Klebsiella, Serratia, Proteus,Campylobacter, Haemophilus, Morganella, Vibrio, Yersinia, Acinetobacter,Stenotrophomonas, Brevundimonas, Ralstonia, Achromobacter,Fusobacterium, Prevotella, Branhamella, Neisseria, Burkholderia,Citrobacter, Hafnia, Edwardsiella, Aeromonas, Moraxella, Brucella,Pasteurella, Providencia, and Legionella. Non-limiting examples ofGram-positive bacteria of this invention include bacteria of thefollowing genera: Enterococcus, Streptococcus, Staphylococcus, Bacillus,Paenibacillus, Lactobacillus, Listeria, Peptostreptococcus,Propionibacterium, Clostridium, Bacteroides, Gardnerella, Kocuria,Lactococcus, Leuconostoc, Micrococcus, Mycobacteria and Corynebacteria.Non-limiting examples of yeasts and molds of this invention includethose of the following genera: Candida, Cryptococcus, Nocardia,Penicillium, Alternaria, Rhodotorula, Aspergillus, Fusarium,Saccharomyces and Trichosporon. Non-limiting examples of parasites ofthis invention include those of the following genera: Trypanosoma,Babesia, Leishmania, Plasmodium, Wucheria, Brugia, Onchocerca, andNaegleria. Non-limiting examples of mollicutes of this invention includethose of the following genera: Mycoplasma and Ureaplasma.

As used herein, the terms “colony” and “microcolony” refer to amultiplicity or population of microorganisms that lie in close proximityto each other, that lie on a surface, and that are the clonaldescendants, by in situ replication, of a single ancestralmicroorganism. In general, a “colony” is visible to the human eye and istypically greater than about 50 μm, 60 μm, 80 μm, or 100 μm, indiameter. However, as used herein, unless otherwise stated, the term“colony” is meant to include both colonies having a diameter of 50 μm ormore, and “microcolonies” having a diameter of 50 μm or less. In otherembodiments, the present invention is directed to scanning, detecting,characterizing and/or identifying microorganisms in a “microcolony.” Asused herein, a “microcolony” can range from about 2 μm to about 50 μm orfrom about 10 μm to about 50 μm. A “microcolony” is generally too smallto be visible to the naked eye (e.g., less than about 50 μm indiameter).

As used herein, the terms “scan” or “scanning” refer to searching apredefined area in a systematic or predetermined pattern, or randomly,to locate something of interest (e.g., a microorganism colony). Forexample, a solid or semi-solid medium can be “scanned” by moving afocused beam of light in a systematic or predetermined pattern, orrandomly, over a surface in order to detect, locate or otherwise sense amicroorganism colony. In an alternative embodiment, the solid orsemi-solid medium can be moved in a systematic or predetermined pattern,or randomly, relative to the light beam to detect, locate or otherwisesense a microorganism colony. In accordance with this embodiment, thelight source typically has a beam diameter of less than about 0.5 mm,less than about 0.2 mm, or less than 0.1 mm. In another embodiment, thebeam diameter is from about 5 μm to about 500 μm, from about 10 μm toabout 100 μm, or from about 20 μm to about 80 μm.

In one embodiment, the “scanning” may comprises a point-by-point “scan”of the solid or semi-solid medium. In accordance with this embodiment alight source (e.g., a laser beam) can be moved to a first point on thesolid or semi-solid medium and a scanning or interrogation step carriedout for the detection and/or characterization of any microorganismcolonies that may be present. Alternatively, the solid or semi-solidmedium can be moved relative to the light source such that apoint-by-point scanning is conducted of the solid or semi-solid medium.Subsequently, the light source (e.g., a laser beam), or the solid orsemi-solid medium, can be moved such that a second point on the mediumcan be scanned and/or interrogated. This point-by-point scanning processcan be continued until a point-by-point search of a given search area iscompleted. The search area can be the entire surface of the solid orsemi-solid medium (e.g., a medium plate) or a subset thereof.

In another embodiment, the point-by-point search can be carried out frompoint-to-point along a linear trajectory (e.g., a long a straight lineacross the medium). Subsequently, the light source, or medium, can beshifted to a second linear line, and a point-by-point search conductedalong the linear trajectory of the second linear line. Thispoint-by-point and line-by-line search pattern (or grid type scan) canbe continue until a given search area is completed. The search area canbe the entire surface of the solid or semi-solid medium (e.g., a mediumplate) or a subset thereof. In another embodiment, the scan be acontinuous scanning (i.e., a continuous point-by-point scanning).

The present invention provides methods for detecting, monitoring,characterizing, and/or identifying microorganisms on a solid orsemi-solid medium. The rapid methods allow the detection,characterization and/or identification of microorganisms more quicklythan prior techniques, resulting in faster diagnoses (e.g., in a subjecthaving or suspected of having an infection), characterization and/oridentification of contaminated materials (e.g., foodstuffs, watersupplies, and pharmaceuticals). The steps involved in the methods of theinvention, from obtaining a sample to characterization/identification ofmicroorganisms, can be carried out in a short time frame to obtainclinically relevant actionable information. In certain embodiments, themethods of the invention can be carried out in less than about 72 hours,e.g., in less than about 18, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 hour.In certain embodiments, the identification steps can be carried out inless than 60 minutes, e.g., less than about 50, 40, 30, 20, 10, 5, 4, 3,2, or 1 minute. The methods can be used to characterize and/or identifyany microorganism as described herein. In one embodiment, themicroorganism is a bacterium. In another embodiment, the microorganismis a yeast. In another embodiment, the microorganism is a mold. Themethods can be used to detect, monitor, characterize, and/or identifymultiple types of microorganisms, e.g., microorganisms of differentspecies, genuses, families, orders, classes, phyla, and/or kingdoms. Inone embodiment, the methods of the invention permit the characterizationand/or identification of some or all of the different types ofmicroorganisms present in a sample, e.g., in a mixed culture. In otherembodiments, the methods can be used to characterize and/or identify twoor more different types of bacteria, two or more different types ofyeast, two or more different types of mold, or two or more differenttypes of a mixture of bacteria, yeast, and or mold. The detection ofeach of the multiple types of microorganisms can occur simultaneously ornearly simultaneously. Additionally, the methods of the invention can bepartially or fully automated, thereby reducing the risk of handlinginfectious materials and/or contaminating the samples.

A first aspect of the invention relates to methods of characterizingand/or identifying a microorganism on a solid or semi-solid medium,comprising:

(a) interrogating one or more colonies on a medium to produce intrinsicfluorescence (IF) measurements characteristic of a microorganism in thecolony; and(b) characterizing and/or identifying the microorganism in the colonybased on the produced measurements.

Another aspect of the invention relates to methods of detecting,characterizing and/or identifying a microorganism on a solid orsemi-solid medium, comprising:

(a) scanning a medium known to contain, or that may contain one or moremicroorganism colonies to locate said colonies present on the medium;(b) interrogating one or more colonies located during step (a) toproduce intrinsic fluorescence (IF) measurements characteristic of amicroorganism in the colony; and(c) characterizing and/or identifying the microorganism in the colonybased on the produced measurements.

A further aspect of the invention relates to methods of characterizingand/or identifying a microorganism in a sample, comprising:

(a) growing a microorganism present in the sample on a solid orsemi-solid medium to produce at least one colony;(b) interrogating one or more colonies on the medium to produceintrinsic fluorescence (IF) measurements characteristic of themicroorganism; and(c) characterizing and/or identifying the microorganism in the colonybased on the produced measurements.

An additional aspect of the invention relates to methods of detectingthe presence of a microorganism in a sample, comprising:

(a) obtaining a sample known to contain or that may contain amicroorganism;(b) growing a microorganism present in the sample on a solid orsemi-solid medium; and(c) locating any colonies present on the medium by scanning the mediumto produce intrinsic fluorescence (IF)measurements;wherein the presence of one or more colonies as located by the producedmeasurements indicates that a microorganism is present in the sample.

Samples that may be tested by the methods of the invention include bothclinical and non-clinical samples in which microorganism presence and/orgrowth is known or suspected, as well as samples of materials that areroutinely or occasionally tested for the presence of microorganisms. Theamount of sample utilized may vary greatly due to the versatility and/orsensitivity of the method. Sample preparation can be carried out by anynumber of techniques known to those skilled in the art.

Clinical samples that may be tested include any type of sample typicallytested in clinical and/or research laboratories, including, but notlimited to, blood, serum, plasma, blood fractions, joint fluid, urine,semen, saliva, feces, cerebrospinal fluid, gastric contents, vaginalsecretions, tissue homogenates, bone marrow aspirates, bone homogenates,sputum, aspirates, swabs and swab rinsates, other body fluids, and thelike.

The present invention finds use in research as well as veterinary andmedical applications. Suitable subjects from which clinical samples canbe obtained are generally mammalian subjects, but can be any animal. Theterm “mammal” as used herein includes, but is not limited to, humans,non-human primates, cattle, sheep, goats, pigs, horses, cats, dog,rabbits, rodents (e.g., rats or mice), etc. Human subjects includeneonates, infants, juveniles, adults and geriatric subjects. Subjectsfrom which samples can be obtained include, without limitation, mammals,birds, reptiles, amphibians, and fish.

Non-clinical samples that may be tested include substances encompassing,but not limited to, foodstuffs, beverages, pharmaceuticals, cosmetics,water (e.g., drinking water, non-potable water, and waste water),seawater ballasts, air, soil, sewage, plant material (e.g., seeds,leaves, stems, roots, flowers, fruit), blood products (e.g., platelets,serum, plasma, white blood cell fractions, etc.), donor organ or tissuesamples, biowarfare samples, and the like. The method is alsoparticularly well suited for real-time testing to monitor contaminationlevels, process control, quality control, and the like in industrialsettings.

The volume of the sample should be sufficiently large to produce one ormore colonies when plated on medium. Appropriate volumes will depend onthe source of the sample and the anticipated level of microorganisms inthe sample. For example, a clinical swab from an infected wound willcontain a higher level of microorganisms per volume than a drinkingwater sample to be tested for contamination, so a smaller volume of swabmaterial will be needed as compared to the drinking water sample. Ingeneral, the sample size can be at least about 50 ml, e.g., 100 ml, 500ml, 1000 ml or more. In other embodiments, the sample can be less thanabout 50 ml, e.g., less than about 40 ml, 30 ml, 20 ml, 15 ml, 10 ml, 5ml, 4 ml, 3 ml, or 2 ml. In certain embodiments, the sample size can beabout 1 ml or less, e.g., about 750 μl, 500 μl, 250 μl, 100 μl, 50 μl,25 μl, 10 μl, 5 μl, 1 μl, 0.5 μl, 0.1 μl, or less. For embodiments inwhich the sample size is large, the sample can be filtered (e.g.,through a filter membrane) and/or concentrated via methods well known inthe art (e.g., centrifugation, evaporation, etc.) to reduce the volumeand/or collect any microorganisms in the sample. Microorganismscollected on a filter membrane can be resuspended and placed on solid orsemi-solid media or the filter membrane can be placed directly onsemi-solid media.

Samples to be tested are placed on a suitable medium and incubated underconditions that are conducive to growth of microorganisms. The mediumcan be selected based on the type(s) of microorganisms known to be orsuspected to be in the sample. Appropriate growth media for differentmicroorganisms are well known to those of skill in the art. The growthmedia can be any medium that provides appropriate nutrients andrestricts movement of the microorganisms (i.e., provides localizedgrowth). In some embodiments, the medium can be a semi-solid medium,such as agar, gelatin, alginate, carrageenan, or pectin. Suitable mediainclude media having different functions that are well known to those ofskill in the art, including without limitation general purpose media,selective media, differential media, and/or chromogenic media. Media canbe selected and/or adjusted such that meaningful measurements (e.g., IFmeasurements) can be obtained. Examples of suitable semi-solid mediainclude, without limitation, A C agar, Acetobacter agar,Acriflavine-ceftazidime agar, Actinomyces agar, Actinomycete isolationagar, Aeromonas isolation agar, Anaerobic agar, Anaerobic blood agar,Anaerobic TVLS agar, APT agar, Ashby's mannitol agar, Aspergillusdifferentiation agar, ASS agar, Aureus agar, Azide blood agar, B.T.B.lactose agar, Bacillus agar, Baird Parker agar, BiGGY agar, Bile esculinagar, Bile esculin azide agar, Bile salts brilliant green starch agar,Bismuth sulfite agar, Blood agar, Blood agar SLMB, BPL agar, Brain heartinfusion agar, Brewer agar, Brilliant green agar, Brilliant green bileagar, Brilliant green phenol red lactose sucrose agar, BROLACIN agar,BROLACIN MUG agar, Brucella agar, BSM agar, Buffered charcoal yeastextract agar, Calcium caseinate agar, Campylobacter selective agar,Candida ident agar, Casein yeast magnesium agar, CASO agar, CATC agar,Cereus selective agar, Cetrimide agar, Chapman Stone agar, China bluelactose agar, Chlamydospore agar, Christensen citrate agar,Christensen's urea agar, Citrate agar, CLED agar, Clostridium agar,Clostridium difficile agar, Coliform agar, Columbia agar, Columbia bloodagar, Corn meal agar, Corn meal peptone yeast agar, CPC-agar, Crampagar, Czapek dox agar, D.T.M. agar, Davis Minimal agar, DCLS agar,Deoxycholate citrate agar, Deoxyribonuclease test agar, DEV ENDO agar,DEV gelatin agar, DEV nutrient agar, Dextrose caseinpeptone agar,Dextrose starch agar, DHL agar, Dichloran rose bengal agar, Diphtheriavirulence agar, DNase test agar with toluidine, E. coli agar, E. coliO157:H7 MUG agar, ECC agar, ECC selective agar, ECD agar, ECD MUG agar,EMB agar, Endo agar, Enterobacter sakazakii agar, Enterococcus faeciumagar, Enterococcus selective agar, Esculin iron agar, Eugonic agar,Fungal agar, Fungobiotic agar, Gassner agar, Gassner lactose agar,Gelatin iron medium, Gelatin salt agar, Germ count agar, Glucosebromcresol purple agar, GSP agar, Hektoen enteric agar, Kanamycinesculin azide agar, Karmali campylobacter agar, KF-streptococcus agar,King agar, Klebsiella selective agar, Kligler agar, KRANEP agar, Kundratagar, Lactobacillus bulgaricus agar, Lactose TTC agar, LB agar, Leifsonagar, Levine EMB agar, Listeria agar, Listeria mono confirmatory agar,Listeria mono differential agar, Listeria selective agar, Litmus lactoseagar, LL agar, LPM agar, LS differential agar, L-top agar, Luria agar,Lysine arginine iron agar, Lysine iron agar, M enterococcus agar, M-17agar, MacConkey agar, MacConkey agar with crystal violet, sodiumchloride and 0.15% bile salts, MacConkey MUG agar, MacConkey-sorbitolagar, Malt agar, Malt extract agar, Mannitol salt phenol red agar,McBride agar, McClung Toabe agar, M-CP agar, Meat liver agar, Membranefilter enterococcus selective agar, Membrane lactose glucuronide agar,M-Endo agar, M-Endo agar LES, MeReSa agar, M-FC agar, Middlebrook 7H10agar, Middlebrook 7H11 agar, Milk agar, Mitis salivarius agar, MM agar,Modified buffered charcoal agar, MOX agar, MRS agar, MS.O157 agar, M-TECagar, Mueller Hinton agar, MUG tryptone soya agar, Mycoplasma agar,Noble agar, Nutrient agar, Nutrient gelatin, OF test nutrient agar, OGYagar, OGYE agar, Orange serum agar, Oxford agar, PALCAM listeriaselective agar, Pentachloro rose bengal yeast extract agar, Peptoneyeast extract agar, Peptonized milk agar, Perfringens agar, Phenol reddextrose agar, Phenol red lactose agar, Phenol red maltose agar, Phenolred sucrose agar, Phenol red tartrate agar, Phenolphthalein phosphateagar, Phenylalanine agar, Plate count agar, Plate count MUG agar, PLETagar, PM indicator agar, Potato dextrose agar, Potato glucose rosebengal agar, Potato glucose sucrose agar, Pril® mannitol agar,Pseudomonas agar, R-2A agar, Raka-Ray agar, Rapid enterococci agar,Reinforced clostridial agar, Rice extract agar, Rogosa agar, Rogosa SLagar, Rose bengal agar, Rose Bengal chloramphenicol agar, S.F.P. agar,Sabouraud 2% glucose agar, Sabouraud 4% glucose agar, Sabouraud dextroseagar, Sabouraud glucose agar with chloramphenicol, Salmonella agar,Salmonella agar according to Oenδz, Salmonella chromogen agar, SD agar,Select agar, Selective agar for pathogenic fungi, SFP agar, S-Gal®/LBagar, Shapton agar, Simmons citrate agar, Skim milk agar, Sorbic acidagar, Spirit blue agar, SPS agar, SS-agar, Standard nutrient agar no. 1,Staphylococcus agar, Streptococcus selective agar, Streptococcusthermophilus isolation agar, Sulfate API agar, Sulfite iron agar, TBXagar, TCBS agar, TCMG agar, Tergitol®-7 agar, Thayer Martin agar,Thermoacidurans agar, Tinsdale agar, Tomato juice agar, Tributyrin agar,Triple sugar iron agar, Tryptic soya agar, Tryptone agar, Tryptoneglucose extract agar, Tryptone glucose yeast extract agar, Tryptone soyayeast extract agar, Tryptone yeast extract agar, Tryptose agar, TSCagar, TSN agar, Universal beer agar, UTI agar, Vibrio agar, Vibrioparahaemolyticus sucrose agar, Violet red bile agar, Violet red bileglucose agar, Violet red bile lactose agar, Violet red bile lactosedextrose agar, Vitamin B₁₂ culture agar, Vogel-Johnson agar, VRB MUGagar, Wilkins Chalgren anaerobic agar, Wilson Blair agar, WLdifferential agar, WL nutrient agar, Wort agar, XLD agar, XLT4 agar,Yeast agar, Yeast extract agar, Yeast malt agar, Yeast mannitol agar,Yersinia isolation agar, Yersinia selective agar, YGC agar, YPAD agar,YPDG agar, YPG agar, and YT agar. In one embodiment, the solid orsemi-solid medium may further comprise one or more additional additivesthat enhance or otherwise increase intrinsic fluorescence (IF)measurements of a microorganism colony on the solid or semi-solidmedium. Suitable additives for enhancing intrinsic fluorescence mayinclude one or more protein hydrolysates, amino acids, meat andvegetable extracts, carbohydrate sources, buffering agents,resuscitating agents, growth factors, enzyme cofactors, mineral salts,metal supplements, reducing compounds, chelating agents,photosensitizing agents, quenching agents, reducing agents, oxidizingagents, detergents, surfactants, disinfectants, selective agents,metabolic inhibitors, or combinations thereof.

In other embodiments, the medium can be a filter (e.g., a filtermembrane or a glass fiber filter), e.g., that is laid on top of asemi-solid medium. In other embodiments, the filter is laid over amaterial (e.g., an absorbent pad) that has been exposed to (e.g., soakedin) liquid medium. In some embodiments, a sample (e.g., a large volumesample) may be passed through a filter to collect any microorganismspresent in the sample. The filter can then be placed on top of growthmedia and incubated under appropriate conditions for microorganismgrowth. Suitable filter membranes are well known to those of skill inthe art and include any membrane suitable for collecting microorganismsand/or capable of supporting microorganism growth. Examples of membranematerials include, without limitation, cellulose, mixed cellulose ester,nitrocellulose, polyvinylchloride, nylon, polytetrafluoroethylene,polysulfone, polyethersulfone, polycarbonate black, and black mixedester, including any combination thereof. The filters can have a poresize suitable for filtering liquids and/or collecting microorganisms,e.g., about 1 to about 25 μm for yeast and about 0.05 μm to about 2 μm,e.g., about 0.2 μm to about 1 μm for bacteria.

In certain embodiments, the medium can be present in a plate, e.g., astandard microbiological agar plate. In some embodiments, the plate canbe a multiwell plate, having, e.g., 2, 4, 6, 8, 12, 24, 32, 48, 64, 96,128, or more wells per plate, for testing of multiple samples. The platecan be made of any suitable material for growing microorganisms, e.g.,polystyrene or glass. The plate optionally has a lid. If theinterrogation of colonies occurs while the lid is in place, the lidand/or the plate can contain at least one area that is transparent to atleast a portion of the ultraviolet, visible light, and/or near infraredspectrum to permit interrogation through the lid and/or plate.

In the methods of the invention, the phrases “growing microorganismspresent in the sample on a solid or semi-solid medium” and “a sample isplaced on a medium” include any manner of contacting the sample with themedium such that microorganisms present in the sample can grow andproduce colonies. In certain embodiments, the sample is placed on thesurface of the solid or semi-solid medium. In other embodiments, thesample may be mixed with the medium in a liquid state and than allowedto solidify (e.g., pour plates) such that any colonies that grow areembedded within the medium. In another embodiment, a sample can be mixedwith dehydrated medium such that the medium is rehydrated and thenallowed to solidify.

In one embodiment, the solid or semi-solid medium is at the bottom of acontainer containing microorganisms suspended in a liquid above themedium. The container can then be manipulated (e.g., centrifuged) toplace the microorganisms on the medium. The liquid can then be removedand the medium incubated for colony growth. For example, a blood samplecan be introduced into a blood culture tube containing a liquid growthmedium and a solid or semi-solid medium at the bottom. The culture tubeis then centrifuged to place the microorganisms on the solid orsemi-solid medium (optionally after red blood cells are lysed), theliquid removed, and the microorganisms grown, detected, and/oridentified according to the methods of the invention.

Once a sample is placed on a medium (e.g., by spreading a liquid sampleon the medium using standard microbiological techniques and/or byplacing a filter membrane on a semi-solid medium), the medium isincubated under conditions suitable for growth of microorganisms presentin the sample. Appropriate conditions are well known to those of skillin the art and will depend on the microorganisms and the medium. Themedium can be incubated at a temperature of about 20° C. to about 50°C., e.g., about 25° C. to about 45° C., e.g., about 37° C. Theincubation time is sufficient for detectable colonies to appear(visually or spectroscopically) and will depend on the microorganism(s),the temperature, the medium, the level of nutrients, and otherconditions that determine growth rate. In some embodiments, theincubation time can be about 12 hours or less, e.g., about 11, 10, 9, 8,7, 6, 5, 4, 3, 2, or 1 hour or less. In certain embodiments, such asunder slow growing conditions or with slow growing microorganisms (e.g.,mycobacteria), the incubation time can be about 12 hours or more, e.g.,about 18, 24, 36, 48, or 72 hours or more. In some embodiments, themedium is incubated in an incubator and the medium is removed from theincubator one or more times and placed in an apparatus to detect and/oridentify any colonies growing on the medium. In other embodiments, themedium can be incubated directly in the apparatus used to detect and/oridentify colonies, e.g., in a temperature-controlled plate holder.

In one aspect, the invention relates to the interrogation of a colony ofmicroorganisms on a solid or semi-solid medium to produce IFmeasurements which identify the microorganism that makes up the colony.In one embodiment, the interrogation is by fluorescence spectroscopy.The interrogation can take place in a non-invasive manner, that is, thecolony can be interrogated while it remains intact on the medium. Inanother embodiment, the plate containing the medium and the colonyremains sealed (e.g., the lid is not removed) throughout theinterrogation. In accordance with this embodiment, the plate, or aportion thereof, may be composed of a material that is transparent tolight (e.g., at least a portion of the near infrared (NIR; 700 nm-1400nm), ultraviolet (UV; 190 nm-400 nm) and/or visible (VIS; 400 nm-700 nm)light spectrum). Examples of suitable materials include, withoutlimitation, acrylic, methacrylate, quartz, fused silica, sapphire, acyclic olefin copolymer (COC) and/or a cyclo olefin polymer (COP) (e.g.,Zeonex® (Zeonex®, San Diego, Calif.)). The ability to detect and/oridentify the microorganisms in a non-invasive manner, optionally coupledwith keeping the plate sealed throughout the identification process, aswell as automating some or all of the procedure, decreases the risksfrom handling microorganisms that are or may be infectious and/orhazardous, as well as the risk of contaminating the sample. Furthermore,the ability to identify microorganisms by direct interrogation withoutfurther processing of the pellet (e.g., suspension and replating and/orother identification assays), greatly increases the speed with whichidentification can be made. In other embodiments, the colony issuspended in a solution and optionally removed from the medium prior tointerrogation. In another embodiment, the colony is suspended in asolution after in situ interrogation and further interrogation is thencarried out. For example, techniques such as latex agglutination testsor automated phenotypic identification tests that can be applied toisolated microorganisms but not a colony of microorganisms on a mediumcan be carried out on the suspended microorganisms.

In some embodiments, the spectroscopy can be used to analyze theintrinsic fluorescence properties of the microorganisms, e.g., aproperty present within the microorganism in the absence of additionalagents, such as stains, dyes, binding agents, etc. In other embodiments,in addition to analyzing IF, the spectroscopy can also be used toanalyze one or more extrinsic properties of the microorganism(s), e.g.,a property that can only be detected with the aid of additional agents.The spectroscopic interrogation can be carried out by any techniqueknown to those of skill in the art to be effective for detecting and/oridentifying one or more intrinsic or extrinsic properties ofmicroorganisms. For example, front face fluorescence (where the excitingand emitted light enters and leaves the same optical surface, and if thesample is generally optically thick, the excitation light penetrates avery short distance into the sample (see, e.g., Eisinger, J., and J.Flores, “Front-face fluorometry of liquid samples,” Anal. Biochem. 94:15(1983)) can be used for identification of microorganisms in pellets.Other forms of measurement, such as epifluorescence, reflectance,absorbance, and/or scatter measurements, can also be employed in thepresent invention.

In one aspect of the invention, the spectroscopy is carried out usingfocusing optics, such as a lens system or a microscope set up to permitobservations in the ultraviolet, visible, and infrared rangefunctionally linked to a spectrophotometer (e.g., using fiber optics).In one embodiment, the medium (e.g., in a plate), is placed on amicroscope stage where it can be interrogated by an excitation source aswell as observed visually (e.g., through the microscope). In oneembodiment, the plate can be manually manipulated to position coloniesfor interrogation, either by moving the plate itself or moving themicroscope stage to which the plate is affixed. In another embodiment,the microscope stage is automatically controlled (e.g., a motorizedstage) such that a plate affixed to the stage can be scanned (e.g., in aset pattern designed to cover the entire section to be scanned). Inanother embodiment, the medium held stationary while a focused lightbeam, such as a laser, is scanned across the medium and the emittedlight is detected by an imaging or non-imaging detector. In a furtherembodiment, the microscope can comprise a plate incubator with atemperature control (e.g., a water bath) so that the plate can remainunder the microscope and be interrogated during incubation of themedium.

In one aspect of the invention, an excitation source is directed at asingle colony to produce IF measurements. The colony can be any size atthe time of interrogation as long as it is sufficiently large for anaccurate measurement to be made. In one embodiment, a colony can beinterrogated when it is undetectable by the human eye. For example, acolony can be interrogated when the colony comprises less than about10,000 microorganisms, e.g., less than about 5000, 1000, 500, 400, 300,200, or 100 microorganisms. In other embodiments, a colony can beinterrogated when the colony is less than about 1000 μm in diameter orless than about 1000 μm in length in its longest dimension (if thecolony is not round). For example, a colony can be interrogated when thecolony is about 900, 800, 700, 600, 500, 400, 300, 200, 100, 50, or 25μm or less. In one embodiment, the excitation beam is smaller indiameter than the colony to be interrogated, such that the entire beamcan be directed at a colony and the medium does not substantiallyinterfere with the IF measurement. In certain embodiments, theexcitation beam has a diameter of less than about 1000 μm, e.g., lessthan about 900, 800, 700, 600, 500, 400, 300, 200, 100, 50, or 25 μm.The size of the excitation beam, as well as the size of the emissionbeam, can be controlled, e.g., with the use of pinholes. In someembodiments, the excitation beam is directed at the center of thecolony. In other embodiments, the excitation beam is directed at otherparts of the colony (e.g., at and/or near the edge) where themicroorganisms may be in a different growth and/or metabolic state thanthose at the center of the colony. In a further embodiment, theexcitation beam can be directed at a certain depth within the colony,e.g., using confocal microscopy.

The colony illumination source, or excitation source, may be selectedfrom any number of suitable light sources as known to those skilled inthe art. Any portion of the electromagnetic spectrum that producesusable data can be used. Light sources capable of emission in theultraviolet, visible and/or near-infrared spectra, as well as otherportions of the electromagnetic spectrum, can be utilized and are knownto those skilled in the art. For example, light sources may be continuumlamps such as a deuterium or xenon arc lamp for generation ofultraviolet light and a tungsten halogen lamp for generation ofvisible/near-infrared excitation. These light sources provide a broademission range and the spectral bandwidth for specific excitationwavelengths may be reduced using optical interference filters, prismsand/or optical gratings, as are well known in the art.

Alternatively, a plurality of narrowband light sources, such as lightemitting diodes and/or lasers, may be spatially multiplexed to provide amulti-wavelength excitation source. For example, light emitting diodesare available from 190 nm to in excess of 900 nm and the sources have aspectral bandwidth of 20-40 nm (full width at half maximum). Lasers areavailable in discrete wavelengths from the ultraviolet to thenear-infrared and can be employed in multiplexing methods well known tothose skilled in the art.

The spectral selectivity of any of the light sources may be improved byusing spectral discrimination means such as a scanning monochromator.Other methods of discrimination may be utilized, as known to those ofskill in the art, such as an acousto-optic tunable filter, liquidcrystal tunable filter, an array of optical interference filters, prismspectrograph, etc., and in any combination. A consideration in selectingthe spectral discriminator takes into account the range of tunability aswell as the level of selectivity. By way of illustration, for example, adiscriminator might utilize the wavelength range of 300-800 nm with aselectivity of 10 nm. These parameters generally determine the optimumtechnology necessary to achieve the tunability range as well as theselectivity.

Typically, the light source results in the excitation of the sample,followed by measurement of the emission of fluorescence from the sampleat predetermined time points or continuously. Similarly, the reflectedlight from interaction of the excitation source with the sample may bemeasured to provide pertinent data for detection and/orcharacterization.

The emission from the sample may be measured by any suitable means ofspectral discrimination, and in some embodiments employs a spectrometer.The spectrometer may be a scanning monochromator that detects specificemission wavelengths whereby the output from the monochromator isdetected by a photomultiplier tube and/or the spectrometer may beconfigured as an imaging spectrograph whereby the output is detected byan imaging detector array such as a charge-coupled device (CCD) detectorarray. In one embodiment, a discriminator allows the observation of thefluorescence and/or scattering signal by a photodetection means (such asa photomultiplier tube, avalanche photodiode, CCD detector array, and/orelectron multiplying charge coupled device (EMCCD) detector array).

The spectroscopic technique is used to obtain measurements that arepreferably provided as Excitation-Emission Matrix (EEM) measurements. Asused herein, EEM is defined as the luminescent spectral emissionintensity of fluorescent substances as a function of both excitation andemission wavelength, and includes a full spectrum or a subset thereof,where a subset may contain a single or multiple excitation/emissionpairs. Additionally, a cross section of the EEM with a fixed excitationwavelength may be used to show the emission spectra for a specificexcitation wavelength, and a cross section of the EEM with a fixedemission wavelength may be used to show the excitation spectra for asample. In one embodiment, multiple EEMs are measured at more than onespecific excitation-emission wavelength pair, e.g., at least at 2, 3, 4,5, 6, 7, 8, 9, 10, 15, 20, 25, 50, or more specific excitation-emissionwavelength pairs. In certain embodiments, the number ofexcitation-emission wavelength pairs measured is sufficient to determinethe exact species of the microorganism, e.g., about 5 to about 30 pairs,e.g., about 10 to about 20 wavelength pairs. In other embodiments, thenumber of excitation-emission wavelength pairs measured is sufficient toat least partially identify the microorganism, e.g., to obtainsufficient useful information for action, e.g., information sufficientto identify a classification group as described below. For example, asuitable number of excitation-emission wavelength pairs to provideuseful information for action, such as a classification group, can beabout 2 to about 8 pairs, e.g., about 3 to about 5 pairs.

According to the invention, control measurements are taken for coloniesof known microorganisms, thus allowing for correlation of measured testdata with characterization of the microorganisms of interest usingvarious mathematical methods known to those skilled in the art. Forexample, the data from samples may be compared with the baseline orcontrol measurements utilizing software systems known to one skilled inthe art. More particularly, the data may be analyzed by a number ofmultivariate analysis methods, such as, for example, GeneralDiscriminant Analysis (GDA), Partial Least Squares Discriminant Analysis(PLSDA), Partial Least Squares regression, Principal Component Analysis(PCA), Parallel Factor Analysis (PARAFAC), Neural Network Analysis (NNA)and/or Support Vector Machine (SVM). These methods may be used toclassify unknown microorganisms of interest into relevant groups basedon existing nomenclature, and/or into naturally occurring groups basedon the organism's metabolism, pathogenicity and/or virulence indesigning the system for monitoring, detecting and/or characterizing theorganism as described previously.

In yet another embodiment, non-spectroscopic measurements from thedetection system, such as detection times and growth rates can be usedto assist in the characterization and/or identification ofmicroorganisms from the colony. Additionally, measurements taken from aphotographic image of the solid or semi-solid media can provide valuableinformation on the characterization and/or identity of themicroorganisms in the colony, such as colony size, shape, color anddensity.

In some embodiments of the invention, characterization and/oridentification of the microorganisms in the colony need not involveidentification of an exact species. Characterization encompasses thebroad categorization or classification of biological particles as wellas the actual identification of a single species. Classification ofmicroorganism from a colony may comprise determination of phenotypicand/or morphologic characteristics for the microorganism. For example,characterization of the biological particles may be accomplished basedon observable differences, such as, composition, shape, size, clusteringand/or metabolism. In some embodiments, classification of the biologicalparticles of interest may require no prior knowledge of thecharacteristics of a given biological particle but only requiresconsistent correlations with empiric measurements thus making thismethod more general and readily adaptable than methods based on specificbinding events or metabolic reactions. As used herein “identification”means determining to which family, genus, species, and/or strain apreviously unknown microorganism belongs to. For example, identifying apreviously unknown microorganism to the family, genus, species, and/orstrain level.

In some instances, characterization encompasses classification modelswhich provide sufficient useful information for action to be taken. Asused herein, the preferred classification models comprise grouping intoone or more of the following: (1) Gram Groups; (2) Clinical Gram Groups;(3) Therapeutic Groups; (4) Functional Groups; and (5) Natural IntrinsicFluorescence Groups.

(1) Gram Groups:

Within the Gram Groups classification, microorganisms may be placed intoone of three broad classification categories based on their Gramstaining reaction and overall size, said groups selected from one ormore of the following: (a) Gram positive microorganisms that stain darkblue with Gram staining; (b) Gram negative microorganisms that stain redwith Gram staining; and (c) yeast cells that stain dark blue with Gramstaining, but are very large rounded cells that are distinguished frombacteria by their morphological characteristics and size.

(2) Clinical Gram Groups:

The Gram Groups may be further divided into several sub-categoriesrepresenting distinguishing morphological features. These sub-categoriescomprise all the relevant clinical information reported by anexperienced laboratory technologist, and thus provide a higher level ofidentification than a positive or negative Gram reaction. Thisparticular classification is very helpful because it eliminates concernsabout relying on the quality of a Gram stain and/or the skill level ofthe technician reading the smear by providing the equivalent clinicallyrelevant information with an automated system. More specifically,subcategories of microorganisms based on this classification model maybe selected from one or more of the following: (a) cocci, which aresmall rounded cells; (b) diplococci, which are two small rounded cellsjoined together; (c) rods, which are rectangular shape; and (d) bacilli,which are rod shaped. Examples of these sub-categories that can beascertained by additional morphological information include: (i) Grampositive cocci; (ii) Gram positive cocci in chains; (iii) Gram positivecocci in clusters (i.e., “grape-like” clusters); (iv) Gram positivediplococci; (v) Gram positive rods; (vi) Gram positive rods withendospores; (vii) Gram negative rods; (viii) Gram negative coccobacilli;(ix) Gram negative diplococci; (x) yeast; and (xi) filamentous fungi.

(3) Therapeutic Groups:

The therapeutic groups comprise multiple microbial species that, whenisolated from particular specimen types, are treated with the same classof antibiotics or mixture of antibiotics (Reference: “Sanford Guide toAntimicrobial Therapy 2008”). In many cases, identity to the specieslevel is not required by the clinician to enable a change from initialempiric therapy to a more targeted therapy because more than one speciescan be treated with the same choice of antibiotic(s). Thisclassification level correctly places these “same-treatment”microorganisms into single therapeutic categories. Examples of thischaracterization level include the ability to distinguish highlyresistant Enterobacteriacae (EB) species from sensitive EB species(Enterobacter spp. from E. coli), or fluconazole-resistant Candidaspecies (C. glabrata and C. kruzei) from sensitive Candida species (C.albicans and C. parapsilosis), and so on.

(4) Functional Groups:

According to the invention, microorganisms may also be placed intoseveral groups based upon a mixture of metabolic, virulence and/orphenotypic characteristics. Non-fermentative organisms may be clearlydistinguished from fermentative ones. Furthermore, microorganism speciesthat produce hemolysins may be grouped separately from non-hemolyticspecies. In some cases, these groups represent broader categories thangenus level (e.g., coliforms, Gram negative non-fermentative rods), someat the genus level (e.g., Enterococcus, Candida), and some with closerto species-level discrimination (e.g., coagulase-negative staphylococci,alpha-hemolytic streptococci, beta-hemolytic streptococci,coagulase-positive staphylococci, i.e., S. aureus).

(5) Natural Intrinsic Fluorescence (“IF”) Groups:

Microorganisms may also be placed into categories based on their naturaltendency to group together by their innate and/or intrinsic fluorescencecharacteristics. Some of these groups may be common to

Therapeutic and Functional Group categories. These groupings maycomprise individual species, such as E. faecalis, S. pyogenes, or P.aeruginosa that have characteristic IF signatures and/or may containsmall groups of organisms with relatively conserved IF signatures suchas the E. coli-K. oxytoca or E. aerogenes and C. freundii groups.

In addition to measuring intrinsic properties of microorganisms (such asintrinsic fluorescence) for identification purposes, the methods of thepresent invention can further comprise the use of additional identifieragents to aid in the identification process. Agents that bind tospecific microorganisms, such as affinity ligands, can be used toseparate microorganisms, to identify a class or species of microorganism(e.g., through binding to a unique surface protein or receptor) and/orto identify a characteristic of the microorganism (e.g., antibioticresistance). Useful identifier agents include, without limitation,monoclonal and polyclonal antibodies and fragments thereof (e.g.,anti-Eap for S. aureus identification), nucleic acid probes, antibiotics(e.g., penicillin, vancomycin, polymyxin B), aptamers, peptide mimetics,phage-derived binding proteins, lectins, host innate immunity biomarkers(acute phase proteins, LPS-binding protein, CD14, mannose bindinglectin, Toll-like receptors), host defense peptides (e.g., defensins,cathelicidins, proteogrins, magainins), bacterocins (e.g., lantibiotics,such as nisin, mersacidin, epidermin, gallidermin, and plantaricin C,and class II peptides), bacteriophages, and fluorescent dyes selectivefor nucleic acids, lipids, carbohydrates, polysaccharides,capsules/slime or proteins, including any combination. If the agent doesnot itself give out a detectable signal, the agent can be labeled toprovide a detectable signal, such as by conjugating the agent to amarker (e.g., visible or fluorescent). Markers include, withoutlimitation, fluorescent, luminescent, phosphorescent, radioactive,and/or colorimetric compounds. The agent can be added to themicroorganisms at any step in the methods of the invention, e.g., whenthe sample is placed on the medium and/or after colonies have beendetected. In some embodiments, the presence and/or amount of the agentin the colony can be determined during interrogation of the colony.Other useful identifier agents include substrates for microbial enzymes,chelating agents, detergents, surfactants, disinfectants (eg. alcohols,bleach, hydrogen peroxide) and toxic compounds (eg. sodium azide,potassium cyanide) and metabolic inhibitors such as cyclohexamide, etc.Similarly, many fluorescent compounds for measuring microbial cellviability, metabolism and/or membrane potential may be used as anidentifier agent in the present invention.

In one aspect of the invention, the method can further comprise a stepof recovering the microorganism(s) from the colony and performingadditional tests. The recovered microorganism(s) can be suspended in asuitable medium, e.g., saline. Once suspended, the microorganism(s) canbe subject to any further tests that are desired, as would be known tothose of skill in the art and as described above. In particular, anytest requiring clean samples of microorganisms can be carried out withthe suspended microorganism(s). In some embodiments, additionalidentification/characterization tests can be performed. Examples ofidentification tests include Vitek 2, amplified and non-amplifiednucleic acid tests (NAT), chromogenic and latex agglutination assays,immunoassays, (e.g., employing labeled primary or secondary antibodiesand/or other ligands), mass spectrometry (e.g., MALDI-TOF massspectrometry) and/or other optical techniques such as infraredspectroscopy (FTIR) or Raman spectroscopy. Additional characterizationtests can also be performed, such as drug resistance, antiobiograms,and/or virulence factors. The additional characterization may be part ofa test that was started during the initial identification steps of themethod. For example, the detection of methicillin resistant S. aureuscan begin by adding fluorescently-labeled penicillin to the sample priorto growth of colonies. The presence and/or amount of bound penicillincan then be determined, e.g., in the colony or in microorganismsrecovered from the colony. In certain embodiments, one or moreadditional tests can be carried out within the same system in which theidentification steps are carried out, e.g., in the same apparatus. Inone embodiment, particular additional tests can be selected from anumber of available tests based on the identification made.

In one aspect of the invention, some or all of the method steps can beautomated. As used herein, the term “automated” means computercontrolled. In one embodiment, the various fluorescence emissiondetection and correlation steps are automated, and the resultinginformation obtained from the methods is automatically used to populatea database. In further embodiments, other steps in the method, such asdetection and/or interrogation of colonies, can also be automated.Automating the steps of the methods not only allows more samples to betested more quickly, it also reduces the risks of human errors inhandling samples that may contain harmful and/or infectiousmicroorganisms and reduces the chances of contaminating the samplesand/or exposing the handler to the samples. In one embodiment, theinvention relates to a system for detecting and/or identifying amicroorganism on a solid or semi-solid medium, the system comprising aspectrophotometer and focusing optics, such as a lens system or amicroscope. In other embodiments, the system further comprises amechanism for scanning the surface of the medium and/or a mechanism forcontrolling the environment of (e.g., incubating) the medium.

One aspect of the invention relates to the detection of a colony on asolid or semi-solid medium. The detection optionally is followed byidentification/characterization of the microorganisms in the colony. Inone embodiment, the medium on which a sample has been placed is manuallyscanned for the presence of colonies. In one embodiment, colonies can bedetected visually with the unaided eye. In other embodiments, coloniescan be detected using a microscope. For example, the medium can beobserved under a microscope while the medium, positioned on themicroscope stage, is manually moved under the microscope objective toscan a portion of the medium for the presence of colonies. The mediumcan be moved by manipulating the medium itself (e.g., moving the platecontaining the medium) or moving the microscope stage on which themedium is placed. In other embodiments, the scanning is carried outautomatically. In one embodiment, a motorized microscope stage can beprogrammed to move the medium under the objective in a search patternacross the surface of the medium such that individual portions of themedium can be observed in turn. In another embodiment, the medium heldstationary while a focused light beam, such as a laser, is scannedacross the medium and the emitted light is detected by an imaging ornon-imaging detector. In one embodiment, the medium can be divided intoequal portions (e.g., about 100, 250, 500, or 1000 μm² or more)corresponding to the dimension of the excitation beam and the microscopestage can be stepped in increments such that each portion is placedunder the objective for interrogation. In another embodiment, the mediumcan be observed on a large scale (e.g., the entire plate or a largefraction thereof (e.g., halves, thirds, quarters, tenths, or less) forcolonies. In either embodiment, the location of colonies can bedetermined based on a map created from the scan of the medium. In oneembodiment, the microscope stage can be programmed to move to eachdetected colony in turn to obtain an IF spectrum of each colony. In oneembodiment, the manual or automatic scanning can be repeated at regularintervals (e.g., every 0.5, 1, 2, 3, 4, 5, 6, 8, 10, or 12 hours ormore) to monitor the appearance and/or growth of colonies. In oneembodiment of the invention, the medium is scanned using visible lightto detect colonies, e.g., colonies that are large enough to be seenunder a microscope. In another embodiment, the medium is illuminatedsuch that an intrinsic property of the colonies (e.g., IF) is detected.Peaks of IF over the background level of the medium indicates thepresence of colonies. For example, a fluorescence map of the medium canbe constructed, e.g., by using a scanning excitation beam (such as alaser) and a simple, non-imaging detector. In another embodiment, largearea imaging using an image capture/acquisition device (e.g., a cameraor scanner such as a CCD linear array scanner, a CCD line-scan camera, aCCD 2D array camera, a laser scanning camera, or other device) can beused as described in WO 03/022999 and U.S. Pat. Nos. 5,912,115,6,153,400, and 6,251,624.

In certain embodiments of the invention, the detection methods can alsobe used to detect the presence of a microorganism(s) in a sample, withor without identification of the detected microorganism. In someembodiments, the detection methods can be used to monitor samples forcontamination by a microorganism, e.g., foodstuffs, pharmaceuticals,drinking water, etc. In one embodiment, the methods can be carried outin a repetitive fashion for constant monitoring for contamination, e.g.,once a month, once a week, once a day, once an hour, or any other timepattern. In another embodiment, samples can be tested as needed, e.g.,when contamination is suspected or absence of contamination needs to beconfirmed. In further embodiments, the detection methods can be used tolook for the presence of a microorganism in a clinical sample, e.g.,from a wound or blood culture. For example, a sample can be removed froma blood culture at certain time points and the detection method carriedout on the sample to determine if the blood culture is positive. In oneembodiment, a sample may be taken at a set time point after inoculationof the culture, e.g., 24 hours after inoculation, to determine if theblood culture is positive. In another embodiment, samples can be takenfrom the blood culture regularly, e.g., every 12, 6, 4, 2, 1, or 0.5hours, to identify positive blood cultures within a short time of beingdetectably positive. In certain embodiments of the detection methods,the detection step can optionally be followed byidentification/characterization methods as described herein. In otherembodiments, the detection methods are partially or fully automated,particularly for the embodiments involving repetitive monitoring ofsamples.

In certain embodiments, the methods of the invention can be carried outwith animal or plant cells instead of microorganisms. In particular,animal cells (e.g., mammalian, avian, insect, cells) or plant cells thatcan grow in colonies, clumps, or other three-dimensional structures orthat are grown on three-dimensional substrates can be detected,monitored, characterized, and/or identified using the techniquesdisclosed herein. Examples of suitable cells that grow inthree-dimensional colonies include, without limitation, stem cells,fibroblasts, and neoplastic cells.

The present invention is further detailed in the following examples,which are offered by way of illustration and is not intended to limitthe invention in any manner. Standard techniques well known in the artor the techniques specifically described below are utilized.

EXAMPLES Example 1 Obtaining Spectra from Colonies on Plates andMembranes

Tests were conducted to determine whether useful spectra could beobtained of colonies directly on blood agar plates (BAP; tryptic soyagar with 5% sheep blood), with and without black membranes (Table 1).Colonies of E. coli (EC), S. aureus (SA), E. faecalis (EF), and P.aeruginosa (PA) were grown as indicated in Table 2 and spectra weretaken through the UV microscope (10× Objective) coupled with a fiberoptic adaptor to a Fluorolog3 spectrometer (Horiba Jobin Yvon, EdisonN.J.) and a PMT detector. The EEM was acquired through a wavelengthrange of Excitation (Ex)=260-550 nm, and Emission (Em)=280-600 nm, every5 nm with a slit width=5 nm. Where indicated, the interrogation area wasnarrowed by placing a 1 mm pinhole in the emission path which resultedin an observed area of approximately 0.1 mm. Without the pinhole, theexcitation and emission circles as projected on the colonies were equalat approximately 1 mm diameter. The samples that were included in eachtest run are indicated in Table 2.

TABLE 1 Test Run A1 A2 A3 A4 B1 B2 Approx Colony 0.4 Blank 0.2-0.351.0-3.0 1.0-3.0 1.0-3.0 Diameter (mm) Membrane Pall All WME None NoneWME EM Beam 0.1 0.1 0.1 0.1 1.0 1.0 Diameter (mm) Integration Time 1  0.5 0.5 0.5 0.1 0.1 (sec) Membranes: None = Sheep Blood Agar (SBA) Pall= Pall Metricel Black gridded Polyethersulfone membrane on SBA WPC =Whatman Track-Etched Polycarbonate Black membrane on SBA WME = WhatmanBlack Mixed Ester membrane on SBA

TABLE 2 A1 EC1 = ATCC 25922 25 hr @ Ambient Temperature (AT) on PallMetricel Black gridded polyethersulfone membrane on SBA plate.Approximate whole colony diameter spanned ~3 grid dots, the pinholecovered approximately ¼-⅕ of the colony diameter, and 1/20 of the colonyarea. A2 Plain BAP and each membrane on BAP A3 EC3 = ATCC 25922overnight (O/N) @ Ambient Temperature (AT) + 4 h @ 36° C. to produce a350 micron diameter colony on WME membrane SA1 = ATCC 25923 O/N @ RT + 4h @ 36° C. to produce a 200 micron diameter colony on WME membrane A4EC2 = ATCC 25922 O/N colony @ 36° C. on BAP SA2 = ATCC 25923 O/N colony@ 36° C. on BAP EF1 = ATCC 29212 O/N colony @ 36° C. on BAP PA1 = ATCC27853 O/N colony @ 36 C on BAP B1 EC1 = ATCC 25922 O/N colony @ 36° C.on BAP, colony = 2.3 mm dia EC2 = ATCC 25922 O/N colony @ 36° C., colony= 2.3 mm dia, Slit width = 3 nm SA1 = ATCC 25923 O/N colony @ 36° C. onBAP, colony = 2.0 mm dia EF1 = ATCC 29212 O/N colony @ 36° C. on BAP,colony = 1.2 mm dia PA1 = ATCC 27853 O/N colony @ 36° C. on BAP, colony= 3.0 mm dia B2 EC1 = ATCC 25922 O/N colony @ 36° C. on WME membrane onSBA, colony dia = 2.0 mm EC2 = ATCC 25922 O/N colony @ 36° C. on WMEmembrane on SBA, colony dia = 1.6 mm SA1 = ATCC 25923 O/N colony @ 36°C. on WME membrane on SBA, colony dia = 1.2 mm SA2 = ATCC 25923 O/Ncolony @ 36° C., colony dia = 1.2 mm, Int. Time = 0.05 sec, Slits = 2.5nm SA3 = ATCC 25923 O/N colony @ 36° C., colony dia = 1.2 mm, Int. Time= 0.1 sec, Slits = 3.0 nm SA4 = ATCC 25923 O/N colony @ 36° C. on WMEmembrane on SBA, colony dia = 1.1 mm SA5 = ATCC 25923 O/N colony @ 36°C. on WME membrane on SBA, colony dia = 1.2 mm EF1 = ATCC 29212 O/Ncolony @ 36° C. on WME membrane on SBA, colony dia = 1.0 mm PA1 = ATCC27853 O/N colony @ 36° C. on WME membrane on SBA, colony dia = 1.1 mm

Spectra from test run A2 of uninoculated plates are shown in FIGS.1A-1D. The vertical axis on each figure indicates the Ex range, and thehorizontal axis shows the Em range. Spectra were obtained from BAP (FIG.1A), Pall membrane (FIG. 1B), WME membrane (FIG. 1C), and WPC membrane(FIG. 1D) without microorganisms. The first observation was thedifference in background fluorescence between the Pall and Whatmanmembranes and the BAP. Surprisingly, the black Pall membrane fluorescedstrongly in the areas of the spectrum previously found to be importantfor classification of microbial suspensions, much more so than theunmasked BAP. However, the WME membrane gave the least backgroundfluorescence of all.

Spectra from test run A3 of colonies on WME membrane over BAP are shownin FIGS. 2A-2C. Spectra were obtained from EC3 (FIG. 2A) and SA1 (FIG.2B), and the result of subtracting the EC3 spectrum from the SA1spectrum is shown in FIG. 2C. The spectra of the colonies show cleardifferences between S. aureus and E. coli. The fact that some parts ofthe spectrum are higher for E. coli and others are higher for S. aureusshows that the differences are present in the overall pattern, and notsimply differences in the scale of intensity.

Spectra from test run B1 of colonies on BAP without a membrane are shownin FIGS. 3A-3D. Spectra were obtained from EC1 (FIG. 3A), SA1 (FIG. 3B),EF1 (FIG. 3C), and PA1 (FIG. 3D). Although the different measurementparameters produce much higher intensities than the A3 spectra, and inspite of being measured directly on a BAP without a black membrane, therelative patterns are still similar for the respective species ofbacteria.

These experiments showed that intrinsic fluorescence spectra could beobtained of colonies through the microscope directly on a BAP, with orwithout the aid of a black membrane to reduce background fluorescence,and the patterns observed were characteristic for different types ofmicroorganisms.

Example 2 Scanning for Microcolonies Through the Microscope

Tests were carried out to determine whether colonies growing under themicroscope on a motorized stage could be located by using point-by-pointIF measurements, and have IF spectra automatically collected of eachcolony detected. A UV microscope was coupled to a Fluorolog3 (HoribaJobin Yvon, Edison N.J.) spectrometer, which served as the fluorescenceexcitation source and the emission measurement device, via fiber opticcables. The microscope's motorized stage was fitted with a compact plateincubator constructed with coils of tubing fed by a circulatingwaterbath set to 36° C. The incubator was also equipped with a UVtransparent window made from a quartz coverslip. Various agar media wereinoculated by spread method with E. coli ATCC 25922 (EC) and/or S.aureus ATCC 25923 (SA) as indicated in Table 3. Some runs used a lightblocking material, either a black Whatman Mixed Ester (WME) membrane orcharcoal, to reduce the fluorescence coming from the media itself.

After inoculation, the microscope's motorized stage was programmed toperiodically move across the agar plate in a search grid and measure thefluorescence at each point with one or more excitation/emissionwavelength pairs. The Fluorolog3 was programmed with slit widths set to10 nm and integration times set to 500 ms (test runs A-E) or 1000 ms(test runs F-H). The excitation beam projected on the surface of theagar was restricted to roughly 0.1 mm diameter by placing a pinhole inthe excitation beam within the microscope. Corresponding with this beamsize, the microscope stage was stepped in 0.1 mm increments so that 10steps covered 1 mm of distance. The emission beam was not restrictedwith a pinhole, but the microscope was shrouded during measurements toprevent any stray light that was not generated by the excitation beamfrom being detected.

For test runs G and H, an algorithm was developed to automaticallycalculate the location of growing colonies. For Run H the program wasfurther enhanced so that all colonies that were detected triggered themicroscope stage to move to their locations in sequence and collecttheir IF spectra. The spectra collected were a subset of a full matrixscan that comprised 300 EEM points selected to reduce the acquisitiontime required. Also for the sake of time, the instrument was programmedto take spectra of no more than 10 colonies.

TABLE 3 Variables for each experimental run Search Run BacteriaWavelengths Area Steps Scan Time Media A EC 305-365 & 59 × 60  87 minWME-SBA 440-525 B EC 305-365 & 68 × 68 115 min WME-SBA 440-525 C EC305-365 71 × 71 125 min TSA w/ 2% Charcoal D EC 440-525 100 × 100 122min TSA w/ 2% Charcoal E EC 305-365 97 × 97 115 min SBA F EC & SA440-525 72 × 73 111 min SBA G EC & SA 305-365 61 × 61  46 min SBA H EC &SA 440-525 70 × 70  61 min SBA

Run A had an instrument problem that stopped the program after 6 h, andno colonies were detected during that time.

Run B showed one colony barely above background fluorescence at 8 h, 2clearly visible at 10 h, and 3 at 12 h and later. The difference betweenthe colony signals and the background was larger at 440-525 nm (roughly4× background) than at 305-365 nm (roughly 2× background).

Run C had no colonies within the scan area due to a low inoculum.

Run D showed 1 colony at 8 h, and 3 at 10 h and later.

Run E had no colonies in the field of view initially. One colony grewinto the field edges by 12 h, 3 at 14 h and later.

Run F with a mixed inoculum showed 10 colonies determined to be EC by 8h, additionally 3 SA by 10 h, and 2 more SA at times greater than 12 h.Three dimensional plots of the point-by-point IF search scans of run Fare shown in FIGS. 5A-5F, where height equals fluorescence intensity.The plots show measurements taken at 6 h where the first detected colonywas observed (FIG. 4A), 8 h (FIG. 4B), 10 h (FIG. 4C), 12 h (FIG. 4D),16 h (FIG. 4E), and 24 h (FIG. 4F). Note that all colonies visible at 8h were E. coli, whereas the additional colonies seen at 10 h and laterwere S. aureus. A close-up image of the BAP from run F after 24 h isshown in FIG. 5A with the search scan area outlined, A contour plot offluorescence intensity from the search scan at 12 h showingcorresponding colony locations is shown in FIG. 5B.

Run G showed 1 EC colony at 8 h and 2 EC colonies at 9 h. An instrumenterror halted acquisition at 10 h and 12 h, but when restarted at 12 hthere were 5 colonies detected; 2 EC and 3 SA. The colony detectionalgorithm successfully identified the location of all 5 colonies.

Run H had condensation on the observation window that interfered withall scans prior to 9 h, at which time 3 EC colonies were detected andspectra taken. The same 3 EC were detected on subsequent scans, and,beginning at 13 h, 4 SA colonies were also detected and spectra taken.

The experimental results show that intrinsic fluorescence can be used todetect the presence and number of microorganism microcolonies while theyare growing directly on agar plates, whether or not a backgroundblocking membrane or charcoal are used. Furthermore, once located, it isrelatively simple to take full spectra of the microcolonies in situ forclassification.

Example 3 Classification of Microorganism Colonies on Agar Plates

Tests were carried out to determine whether microbial colonies could beclassified from IF spectra taken directly on the agar plate where theywere grown.

The spectral acquisition was done across an excitation (Ex) and emission(Em) matrix of wavelengths 260-580 nm and 260-680 nm, respectively, in asubset of 300 EEM points selected to reduce the acquisition timerequired. Additionally, all reflectance wavelengths (where Ex=Em) werealso read. For fluorescence, the slit widths were set to 5 nm bandpassand the integration time was 1000 ms. Each 300 point acquisition tookapproximately 8.1 min to complete.

Table 4 lists the microorganisms tested, which comprised 6 isolates eachof 20 species for a total of 120 tests. Where used to indicate groupingsother than by species, the term Clinical Gram (ClinGram) refers to theclassification level possible by a highly skilled observer reading aGram stain, not just positive, negative or yeast (Table 5). For example,Staphylococci are Gram positive cocci in clusters, while manyStreptococci are Gram positive cocci in chains.

TABLE 4 Gram Negative Gram Positive Yeast A. baumanii, 6 isolates S.aureus, 6 isolates C. tropicalis, 6 isolates E. aerogenes, 6 isolates S.epidermidis, 6 isolates C. glabrata, 6 isolates E. cloacae, 6 isolatesS. pneumoniae, 6 isolates C. albicans, 6 isolates E. coli, 6 isolates E.faecium, 6 isolates K. pneumoniae, 6 isolates E. faecalis, 6 isolates P.aeruginosa, 6 isolates S. pyogenes, 6 isolates S. maltophilia, 6isolates S. agalactiae, 6 isolates S. marcescens, 6 isolates B.subtilis, 6 isolates B. cereus, 6 isolates

Table 5 shows the results of classification modeling by Forward StepwiseLinear Discriminant Analysis with “Leave-one-out” cross-validation.“Leave-one-out” cross-validation was chosen because it efficiently makesuse of small data sets, estimating the results as if a number of“unknowns” were tested equal to the “training” set, without needing torun twice as many tests. In the tables, the “Number of DA steps” refersto the number of Discriminant Analysis steps completed, which may or maynot be the actual number of EEM points used to produce the indicatedresults. Typically the step number is equal to the number of ExEm pointsin the model, but sometimes the number of model points is less if pointswere removed, rather than added, during some steps.

Since Discriminant Analysis can “find” false correlations in the randomfluctuations within the data, given a sufficient number of sufficiently“noisy” data points, cross validation is essential to estimate the truesuccess of a given classification model. Generally, thenon-cross-validated results will trend to 100% correct with increasingstep count, while the cross-validated results will rise to a peak, andthen tail back down. The model with the number of steps near thecross-validation peak can be considered optimized for a given data set.Table 5 shows the results of each classification model run, both withand without cross validation, at the point where the cross validatedresults are optimized.

The classification for each microorganism by discriminant analysis wasconsidered to be correct if the model's first choice for classificationwas the actual identity of the microorganism, regardless of how closethe other choices may have been. Also shown is the is number andpercentage of microorganisms for which the actual identity is within thetop 3 choices of the model's classification, indicating a goodpredictive, if as yet imperfect, model for classification.

The spectra from colonies clearly show the potential to classifymicroorganisms. There is probably some noise in the data, as indicatedby the fact that the results are improved by binning 2 adjacent ExEmpoints. Two known factors contributing to the noisy data come frominconsistent positioning of the measurement beam on the colonies, andthe low light levels reaching the detector through the apparatus. Forthis study, positioning the excitation beam on the center of thecolonies with the microscope camera was difficult because the lightingavailable for visualization was not optimal. In fact, the positioningwas observed to be off on some occasions, which was corrected, but it islikely that other mis-positionings went unnoticed. Noise in thefluorescence signal itself was also large because the amount offluorescent energy reaching the detectors was more than 1000 times lowerthan for suspensions of microorganisms. This was because of the fiberoptic and microscope configuration, which is a flexible research toolbut is not optimized for this type of measurement. An optical systemdesigned for this task could easily overcome this issue.

TABLE 5 Number (%) Number (%) Number (%) EEM Classification Number ofCorrect w/o Correct with Within Top3 with Points Grouping DA Steps CrossValidation Cross Validation Cross Validation Binned Species 30 118 98.3%86 71.7% 108 90.0% 1 Species 18 113 94.2% 94 78.3% 114 95.0% 2 ClinGram18 118 98.3% 112 93.3% 118 98.3% 2

Example 4 Improved Classification of Colonies with Less Noise

Tests were conducted to determine whether better positioning andincreased light throughput could improve the classification of microbialcolonies with intrinsic fluorescence. The experiment of Example 3 wasrepeated with the same equipment and the same microorganism strains, butwith a modified method. The spectral acquisition was done across thesame Excitation (Ex) and Emission (Em) wavelength range (Ex=260-580 nm,Em=260-680 nm) but with a different subset of 312 wavelengths that coverthe same key areas of the spectrum, but is more conducive to binning ofvalues than was the previous subset. The main reason for using subsetsis to reduce the time required to collect spectra with the currentequipment. The monochromator slit widths were widened to 7 nm bandpassover the previous 5 nm, which increased the measured fluorescence byroughly 2-fold. The integration time was kept at 1000 ms, and eachacquisition took approximately 9.8 min to complete.

Table 6 shows the results of classification modeling by Forward StepwiseLinear Discriminant Analysis with “Leave-one-out” cross-validation. Asbefore, the classification was considered correct if the model's firstchoice for classification was the actual identity of the microorganism,regardless of how close the other choices may have been. Classificationperformance to the species level is shown based on individual datapoints (no binning), binning 3 adjacent fluorescence readings in an “L”pattern on the ExEm matrix, and binning of 4 adjacent EEM points in asquare. Also, classification to the Clinical Gram level is shown with“3L” binning.

The method changes to improve the consistency of the fluorescencereadings combined with the increased light throughput substantiallyimproved the classification success. Of the main improvements, betterpositioning probably contributed more to the performance gain than theincreased light throughput, and it is likely that fluorescence readingsfrom more than one location in each colony could be used to furtherenhance classification accuracy. Limitations in the current equipmentpermitted only a modest 2-fold increase in signal without reducingspectral resolution or increasing the scan time substantially, and it isevident that there is still significant read noise in the fluorescencespectra.

The read noise is partially overcome by binning adjacent points, whichhas no positive effect on other factors affecting classificationsuccess, but reduces the spectral resolution accordingly. That binninghelps also indicates that some spectral resolution might be sacrificedto improve read noise in an optimized system. The improvement withbinning, however, was not as large as the difference between these dataand the results of the previous method, which probably shows thatpositioning of the measurement played a larger role. Furtherimprovements in classification success could be made with automatedpositioning and optimized optics as would be well within the skill ofthe ordinary artisan.

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein. Allpublications, patent applications, patents, patent publications, and anyother references cited herein are incorporated by reference in theirentireties for the teachings relevant to the sentence and/or paragraphin which the reference is presented.

TABLE 6 Number (%) Number (%) Number (%) EEM Classification Number ofCorrect w/o Correct with Within Top3 with Points Grouping DA Steps CrossValidation Cross Validation Cross Validation Binned Species 28 120 100%105 87.5% 116 96.7% 1 Species 40 120 100% 109 90.8% 113 94.2% 3 Species40 120 100% 107 89.2% 117 97.5% 4 ClinGram 28 120 100% 119 99.2% 120100.0% 3

1. A method of detecting and characterizing a microorganism on a solidor semi-solid medium, comprising: (a) scanning a solid or semi-solidmedium, known to contain, or that may contain one or more microorganismcolonies to locate any colonies present on the medium; (b) interrogatingone or more of said colonies located during step (a) wherein theexcitation beam is smaller in diameter than the colony to beinterrogated, to produce intrinsic fluorescence (IF) measurementscharacteristic of a microorganism in said colony; and (c) characterizingthe microorganism in the colony based on said intrinsic fluorescence(IF) measurements, wherein said microorganism is characterized into onone or more classification models selected from the group consisting ofGram Groups, Clinical Gram Groups, Therapeutic Groups, FunctionalGroups, and Natural Intrinsic Fluorescence Groups.
 2. The method ofclaim 1, wherein said scanning comprises a point-by-point scanning ofthe surface of said solid or semi-solid medium.
 3. The method of claim1, wherein said colony is a microcolony having a diameter of less than50 μm.
 4. The method of claim 1, wherein said interrogation step isnon-invasive. 5-6. (canceled)
 7. The method of claim 1, wherein said IFmeasurements are produced by spectroscopy and said spectroscopycomprises determining an excitation-emission matrix (EEM).
 8. The methodof claim 7, wherein said EEM comprises at least two different wavelengthpairs.
 9. The method of claim 7, wherein said EEM is compared to adatabase of EEMs of known microorganisms.
 10. The method of claim 1,further comprising addition of an identifier agent to the medium orsample and wherein the characterization is based in part on the presenceand/or amount of said identifier agent in the colony or inmicroorganisms recovered from the colony.
 11. The method of claim 10,wherein said identifier agent is an affinity ligand, antibody orfragment thereof, nucleic acid probe, antibiotic, aptamer, peptidemimetic, phage-derived binding protein, lectin, host defense peptide,bacterocin, bacteriophage, dye, or any combination thereof.
 12. Themethod of claim 1, wherein said solid or semi-solid medium comprises oneor more nutrients useful for the growth of said microorganism and one ormore additives, wherein said one or more additives enhance saidintrinsic fluorescence measurements of said microorganism colonies onsaid solid or semi-solid medium.
 13. The method of claim 12, whereinsaid one or more additives are selected from the group consisting ofprotein hydrolysates, amino acids, meat and vegetable extracts,carbohydrate sources, buffering agents, resuscitating agents, growthfactors, enzyme cofactors, mineral salts, metal supplements, reducingcompounds, chelating agents, photosensitizing agents, quenching agents,reducing agents, oxidizing agents, detergents, surfactants,disinfectants, selective agents and metabolic inhibitors.
 14. The methodof claim 1, wherein interrogating a colony comprises measuringepifluorescence.
 15. The method of claim 1, wherein interrogating acolony comprises measuring reflected light.
 16. A method of detectingthe presence of a microorganism in a sample, comprising: (a) obtaining asample known to contain or that may contain a microorganism; (b) growingany microorganisms present in the sample on a solid or semi-solidmedium; and (c) locating any colonies present on the medium byconducting a point-by-point scanning of said solid or semi-solid mediumto produce intrinsic fluorescence (IF) measurements; wherein thepresence of one or more colonies as located by the produced measurementsindicates that a microorganism is present in the sample.
 17. The methodof claim 16, wherein said IF measurements are produced by spectroscopyand said spectroscopy comprises determining an excitation-emissionmatrix (EEM).
 18. The method of claim 17, wherein said EEM comprises atleast two different wavelength pairs. 19-34. (canceled)